IL-8, IL-12 and IL-10 cytokines generation by neutrophils, fibroblasts and neutrophils- fibroblasts interaction in psoriasis

Vol. 55(2) · 2010 · pp 254-260 · DOI: 10.2478/v10039-010-0037-0 · Advances in Medical Sciences© ·Medical University of Bialystok, Poland

IL-8, IL-12 and IL-10 cytokines generation by neutrophils, fibroblasts and neutrophils- fibroblasts interaction in psoriasis Glowacka E1, Lewkowicz P1, Rotsztejn H2*, Zalewska A3 1 Department of Clinical Immunology, Polish Mother’s Memorial Hospital - Research Institute, Lodz, Poland 2 Department of Cosmetology, Medical University of Lodz, Lodz, Poland 3 Department of Dermatology and Venerology, Medical University of Lodz, Lodz, Poland

* CORRESPONDING AUTHOR: Department of Cosmetology Medical University of Lodz, 1 Muszynskiego Street, 90-151 Lodz, Poland Tel: 0048604180044; Fax: 0048 42 6779114 e-mail: [email protected] (Helena Rotsztejn)

Received 03.11.2009 Accepted 06.07.2010 Advances in Medical Sciences Vol. 55(2) · 2010 · pp 254-260 DOI: 10.2478/v10039-010-0037-0 © Medical University of Bialystok, Poland

ABSTRACT Purpose: Psoriasis is a common skin disease affecting about 1-3% of the world population. Many types of cells, including lymphocytes, dendritics APCs (antigen presenting cells), NKT (natural killer T) cells, neutrophils, keratinocytes and fibroblasts are involved in the pathogenesis of psoriasis. The aim of our study was to assess in psoriatic patients the production of IL-8, IL-10 and IL-12 cytokines by neutrophils, fibroblasts and fibroblasts - neutrophils interaction. Material/Methods: The production of IL-8, IL-10 and IL-12 cytokines was evaluated in supernatants after cells incubation for 21 h in culture medium alone and in medium in the presence of IL-8 or TNF-α. Concentrations of IL-8, IL-10, IL-12 were measured by enzyme-linked immunosorbent assay (ELISA) method using commercially available kits. Results: Our results demonstrate that fibroblasts are not able to produce IL-10 and IL-12 but they generate IL-8. The amount of IL-8 depends on the site of derivation of fibroblasts and on the stimuli. Neutrophils released IL-8, IL-12 (at a lower level in psoriatic patients) and IL-10 but only in the case of healthy donors and at a very low concentration. Moreover, we observed higher concentrations of IL-12 and IL-8 in supernatants as a result of fibroblasts-neutrophils interaction in psoriatic patients. Conclusions: Our results suggest that fibroblasts take part in the inflammatory reaction in psoriasis via cytokines or direct interaction with neutrophils. Fibroblasts probably do not exert any anti-inflammatory effect. Key words: psoriasis, neutrophils, fibroblasts, cytokines secretion

INTRODUCTION Psoriasis is a chronic inflammatory skin disorder. A combination of genetic, environmental, and immunological factors may be involved in the initiation and development of the disease. Approximately 1-3% of the world population is affected by psoriasis [1]. Abnormal keratinocyte differentiation and proliferation resulting in the formation of typical scaly plaques are characteristic features of psoriasis. Hypotheses of psoriasis pathogenesis have changed over the decade. Recently many investigators have assumed that both innate and adaptive immunity are involved in psoriasis [2,3,4]. In fully developed psoriatic skin lesions there is a mixture of innate immune cells: neutrophils, dendritic APCs (antigen presenting cells)

and NKT (natural killer T) cells and adaptive immune T cells [5,6]. Both adaptive and innate immunity share common effector molecules, particularly cytokines and chemokines. These mediators are crucial for cell-cell interaction, activation and migration of effector cells and the generation of an effective immune response. Based on these data, a novel concept has emerged that the activation of the innate immune system leads to the production of proinflammatory cytokines and aberrant activation of T cells, which in turn release cytokines, leading to psoriatic skin lesions development. The participation of stromal cells in the transition from innate to adaptive immunity has recently been taken into consideration. It means that fibroblasts are not passive players in the immune system, but actively define the structure of tissue and modulate immune

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cell behavior by conditioning the local cellular and cytokine microenvironment [7]. To explain the pathogenesis of psoriasis many investigators have hypothesized the important role of cooperation between different type of cells via cells-cells interaction or via cytokines network. In our study we have assessed the production of IL-8, IL-10 and IL-12 cytokines by psoriatic fibroblasts and neutrophils and the generation of these cytokines as a result of fibroblastsneutrophils interaction.

MATERIAL AND METHODS The study group consisted of 10 psoriasis vulgaris patients (6 males and 4 females), aged 25-45 years, hospitalized at the Department of Dermatology and Venereology, Medical University of Lodz. The control group comprised 10 healthy volunteers (5 females and 5 males) aged 22-46 years. The clinical severity of the disease was evaluated with the psoriasis area and Severity Index PASI score 6.5 – 25.8 [8,9]. In psoriasis vulgaris patients, a 3 mm punch biopsy was taken from a representative psoriatic lesion located on the external surface of the forearm from a plaque centre and from the perilesional uninvolved skin situated about 4-5 mm from a psoriatic plaque edge visible to the naked eye. In the control group, biopsies were taken once from the normal skin of the forearm.

Preparation of neutrophils Blood was collected into pyrogen free Li-heparin tubes in the morning. Neutrophils were separated using density gradient medium Polymorphprep (d = 1.113 ± 0.001 g/ml, AXIS-SHIELD, Norway). Cells were washed three times PBS (Biomed, Lublin, Poland) and resuspended in culture medium (Eagle’s medium (Biomed, Lublin, Poland) supplemented with 20% FCS, 100U/ ml penicillin and 100 μg/ml streptomycin Sigma Cemiacl Co St. Louis, USA). Purity and viability assessed microscopically was ≥ 95%. Then neutrophils were incubated in 24-cells plates (Nunc Corp. Denmark) in culture medium alone, in culture medium with IL-8 (0.1μg/ml) or TNF (10ng/ml) ( Sigma Chemical Co,USA) 21h at 37˚C in 5% CO2 atmosphere. Then supernatants were collected by centrifugation and stored at -80˚C. Concentrations of IL-10 and IL-8 were assessed by ELISA method, using Human IL-8 (sensitivity 1pg/ml) and IL-10 (sensitivity 2pg/ml), ELISA SET BD Opt EIA (San Diego, USA). Concentration of IL-12 in supernatants was assessed by ELISA method using commercially available Endogen Total Human IL-12 (sensitivity<5pg/ml) ELISA Kit (Pierce Endogen; USA). The samples absorbance was read on ELx808 reader at the wave length of 450 nm.

Preparation of fibroblasts Fibroblasts were isolated from 3 mm punch biopsies from involved and uninvolved areas on the external surface of the forearm of ten patients with psoriasis and from agematched controls. The fibroblasts were isolated and cultured as described [10]. For experiments fibroblasts of passage no 3 to 4 were used. Viability determined by trypan blue was ≥ 95%. For studies assessing IL-8, IL-10 and IL-12 secretion by fibroblasts, 105 cells /ml were plated in 24-well plates and incubated in cultured medium alone (Eagle’s medium supplemented with 20% FCS, 100 U/ml penicillin and 100μg/ ml streptomycin) and in culture medium with IL-8 (0.1μg/mlhuman recombinant, Sigma Chemical Co St. Louis, USA and with TNF (10ng/ml-human recombinant, Sigma Chemical Co St. Louis, USA) 72h at 37˚C in 5% CO2 atmosphere. Then supernatants were collected by centrifugation and stored at -80˚C. For assessing IL-10, IL-12 and IL-8 secretion as a result fibroblasts-neutrophils interaction, fibroblasts 105/ml were first preincubated for 6 hours in culture medium alone, in culture medium with IL-8 (0.1μg/ml) or in culture medium with TNF (10ng/ml). After that, 106 neutrophils were added. Cells were incubated for 21 h in 37˚C and in 5% CO2. Then supernatants were collected by centrifugation and stored at -80˚C. Concentrations of IL-10 and IL-8 were assessed by ELISA method, using Human IL-8 (sensitivity 1pg/ml) and IL-10 (sensitivity 2pg/ml), ELISA SET BD Opt EIA (San Diego, USA). Concentration of IL-12 in supernatants was assessed by ELISA method using commercially available Endogen Total Human IL-12 (sensitivity<5pg/ml) ELISA Kit (Pierce Endogen; USA). The samples absorbance was read on ELx808 reader at the wave length of 450 nm. All experiments were repeated twice. All samples were run in duplicate. All the patients and individuals participating in the study gave their informed consent according to the Medical University of Lodz Bioethics Committee requirements.

Statistical analysis The arithmetic means  standard deviation (SD) and standard error of mean (S.E.M.) was calculated for each examined group. Statistical significance of differences among the groups: psoriasis vulgaris patients vs. control group healthy volunteers was determined by the t-Student test (for parametric distribution and equal variances), Cochran Q test (for parametric distribution and unequal variances) or WaldWolfowitz runs (for non parametric distribution). Significant differences between cells alone vs. mixed culture and non- vs. stimulated cultured were determined by using the t-Student test for dependent samples. The verification of parametric distribution and analyses of variances were made using the Kolmogorov-Smirnov test and the Fisher’s test. P0.05 was considered as the significant difference.

IL-8, IL-12 and IL-10 cytokines generation by neutrophils, fibroblasts and neutrophils- fibroblasts interaction in psoriasis

Table 1. IL-8 secretion by neutrophils, fibroblasts and neutrophils – fibroblasts interaction. Cells/Interactions

Unstimulated

Stimulated with TNF-α

NNC

2256.7 ± 363.5

5683.0 ± 618.5*

NPV

2189.0 ± 163.1

5578.0 ± 819.5*

FNC

3783.0± 814.1

302775.0 ± 46524.2 *

FPL

2898.0 ±780.5a

FPPL

1928.0 ± 766.9

FNC + NNC

26993.0 ± 7285.7

FPL + NPV

42376.0± 19368.1e,f,i

418143.0 ± 38590.1 e,f,i

FPPL + NPV

28354.0 ± 15940.2g

489460 ± 174090.8 g,h

274955.0 ± 46592.9 *a 144136.0 ± 36128.7*b

b

507670 ± 93744.4c,d*

c,d

g,h

Neutrophils of normal controls- NNC Neutrophils of psoriatic vulgaris patients –NPV Fibrolasts of normal controls –FNC Fibroblasts from psoriatic lesions –FPL Fibroblasts from psoriatic perilesional –FPPL Results are expressed as mean ± SEM *a,b,c,d,e,f,g,h,i,-p<0.05 *-unstimulated cells vs stimulated cells a- FNC vs FPL; b- FNC vs FPPL; c- FNC+NNC vs NNC; d- FNC+NNC vsFNC; e- FPL+ NPV vs NPV; f- FPL+ NPV vs FPL; g-FPPL+ NPV vs NPV; h- FPPL+NPV vs FPPL; i-FPL+NPV vs FNC +NNC

RESULTS

Fibroblasts

The results illustrating the secretion of IL-8, IL-10 and IL-12 cytokines by neutrophils of peripheral blood of psoriatic patients and of healthy donors, fibroblasts derived from psoriatic lesional skin, fibroblasts from psoriatic perilesional skin and fibroblast normal control skin are presented in Tab. 1, Tab. 2, Tab. 3. Since interleukin IL-8 was used as a stimulator, the secretion of IL-8 was assessed by unstimulated cells and cells treated with TNF.

Fibroblasts, both derived from psoriatic lesional and normal control skin (healthy volunteers), were not able to produce IL-10 and IL-12 with or without IL-8 and TNF-α. We observed a higher level of IL-8 secretion by the normal skin fibroblasts of healthy donors than by fibroblasts derived from psoriatic lesional or psoriatic perilesional area using conditions without or with TNF-α (p <0.05). Both normal fibroblasts, psoriatic fibroblasts and fibroblasts from perilesional area produced IL-8 and the secretion was upregulated by TNF- α (p<0.0001).

Neutrophils The releasing of IL-8 by unstimulated neutrophils of blood in the healthy group was similar to the blood in psoriatic patients (2256±363.5 vs 2188.8±163.0 p>0.05). The generation of IL-8 both by neutrophils of psoriatic patients and healthy donors was modulated using as stimulator TNF-α (p<0.001);(Tab. 1). Both neutrophils of healthy donors and psoriatic patients were able to produce IL-12, but in case of psoriatic patients at a much lower level (p<0.00001). Supplementation culture medium with IL-8 and TNF-α did not modulate secretion of IL-12 either by neutrophils of healthy donors or psoriatic patients (Tab. 2). The secretion of IL-10 was observed at a low, but detectable level only in the case of neutrophils of the blood of healthy donors both by unstimulated (8.5±2.9) and treated with IL-8 (13.8±4.3) and TNF- α (21.4±7.8). As we can see the presence of IL-8 and TNF-α caused an increased release of IL-10 (p<0.05);(Tab. 3)

Fibroblasts-neutrophils interaction We observed: - a higher concentration of IL-12 in conditioning medium as a result of the interaction of fibroblasts derived from psoriatic plaque and from perilesional area with neutrophils of psoriatic in comparison to secretion by neutrophils alone in psoriatic patients (p<0.0001), - a higher concentration of IL-10 in conditioning medium as a result of the interaction of normal control fibroblasts with neutrophils of normal control subjects in comparison to generation by neutrophils alone in group of healthy subjects (p<0.05), - a higher concentration of IL-8 in conditioning medium as a result of the fibroblasts-neutrophils interaction in comparison to secretion by neutrophils alone and fibroblasts alone, both in psoriatic patients (p<0.001) and normal controls p<0.001, however, a slightly higher production was observed in psoriatic patients (p=0.027).

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Table 2. IL-12 secretion by neutrophils, fibroblasts and neutrophils – fibroblasts interaction. Cells/Interactions

Unstimulated

Stimulated with IL-8

Stimulated with TNF-α

NNC

65.1±15.91

59.9±14.72

71±17.11

NPV

10.3±2.12a

15.4±3.3a

12.4±2.34a

FNC

< 5.0

< 5.0

< 5.0

FPL

< 5.0

< 5.0

< 5.0

FPPL

< 5.0

< 5.0

< 5.0

FNC+ NNC

79.9±24.11

82.1±30.12

77.9±24.05

FPL+ NPV

74.5±32.22b

76.7±3.81b

FPPL+NPV

40.3±13.82

53.9±18.15

c

67.6±27.17b c

42.3±14.31c

Neutrophils of normal controls- NNC Neutrophils of psoriatic vulgaris patients –NPV Fibrolasts of normal controls –FNC Fibroblasts from psoriatic lesions –FPL Fibroblasts from psoriatic perilesional –FPPL Results are expressed as mean ± SEM a,b,c-p<0.05 a- NNC vs NPV b-FPL+NPV vs NPV c-FPPL+NPV vs NPV Table 3. IL-10 secretion by neutrophils, fibroblasts and neutrophils - fibroblasts interaction. Cells/Interactions

Unstimulated

Stimulated with IL-8

Stimulated with TNF-α

NNC

8.5 ± 2.93

13.8 ± 4. 1*

21.4 ± 7.82*

NPV

< 2.0a

< 2.0a

< 2.0a

FNC

< 2.0

< 2.0

< 2.0

FPL

< 2.0

< 2.0

< 2.0

FPPL

< 2.0

< 2.0

< 2.0

FNC+ NNC

21.7±6.71b

23.2±7.74 b

28.5±8.93 b

FPL+ NPV

< 2.0

< 2.0

< 2.0

FPPL+NPV

< 2.0

< 2.0

< 2.0

Neutrophils of normal controls- NNC Neutrophils of psoriatic vulgaris patients –NPV Fibrolasts of normal controls –FNC Fibroblasts from psoriatic lesions –FPL Fibroblasts from psoriatic perilesional –FPPL Results are expressed as mean ± SEM *a,b-p<0.05 *-unstimulated cells vs stimulated cells a- NNC vs NPV; b- FNC+NNC vs NNC

DISCUSSION As it has been mentioned in the introduction, in fully developed psoriatic skin lesions we can find a mixture of innate and adaptive immune cells. The development of psoriatic plaque depends on complex interactions among CD4+T cells, CD8+ T cells, NKT cells, APC cells, such as Langerhans cells, plasmocytoid dendritic cells (DCs), mature myeloid DC subsets, neutrophils and mast cells [6]. However, whether the primary cellular target within psoriatic plaques are the macrophages, dendritic cells, T cells, mast cells, neutrophils, keratinocytes, fibroblasts or endothelial cells is still not known. The initial stimulus activating the immune system has not been elucidated either. According to the current knowledge many

cytokines and chemokines contribute to the initiation and maintenance of psoriatic plaques [4,11-16]. We can imagine that the inappropriate mediation by triggering factors may lead to up-regulation of cells of the skin immune system and disturbances in cytokines and chemokines network. One of the earliest pathological changes that occur in psoriatic skin lesions is the appearance of T cell and macrophages. Chemoatraction of neutrophils within the epidermal compartment follows the influx of lymphocytes. In nearly every stage of the disease, neutrophils are present in the epidermis and papillary dermis. The final step in the pathogenesis of psoriasis involves the stimulation and proliferation of keratinocytes by various factors which in turn produce chemokines [17].

IL-8, IL-12 and IL-10 cytokines generation by neutrophils, fibroblasts and neutrophils- fibroblasts interaction in psoriasis

Fibroblasts were previously considered as important connective tissue cells that construct a supporting lattice crucial for tissue integrity and repair. Today we know that fibroblasts can synthesize a number of cytokines, particularly upon stimulation with IL-1 and TNF-α. Moreover, fibroblasts regulate the behaviour of haemopoetic cells that infiltrate damaged tissue through CD40-CD40L interactions [18-20]. There are many results which indicate that dermal fibroblasts may contribute to the epidermal overgrowth of psoriasis by promoting keratinocyte proliferation [21-23]. The hallmark cytokines in a psoriatic network are IL-8 and TNF-α. TNF-α is a key cytokine in the innate immune responses and is increased in psoriatic lesions. Since varieties of other inflammatory cytokines are present in psoriatic lesions, other cytokines can synergize with TNF-α to amplify its inflammatory effects at the local level in the skin. Thus, this single cytokine has the potential for broad-reaching effects in the psoriasis inflammatory cascade [4,12]. IL-8 was initially isolated in large amounts from psoriatic scale. IL-8 and IL-8 R can be induced in many cells by proinflammatory stimuli especially IL-1α, TNF-α, LPS and viral infection. IL-8 and its receptor are greatly increased in psoriatic epidermis and are hypothesized to mediate epidermal hyperproliferation, angiogenesis, and attraction of polymorphonuclear leukocytes and T cells. One hypothesis is that psoriasis involves a final common pathway mediated by proinflammatory cytokines including IL-1, IL-6, and IL-8. IL-8 is increased in psoriatic lesions and stimulates epidermal proliferation [24]. These findings suggest that IL-8 and TNF- α act as psoriasis-promoting factors. For these reasons, in our study we have used IL-8 and TNF- α as potential modulators of secretion of cytokines by neutrophils and fibroblasts of psoriatic patients. We have showed that neutrophils of psoriatic patients secrete equal levels of IL-8 as in normal controls and that this secretion increases after the stimulation with TNF-α. Several previous studies have indicated that neutrophils can release inflammatory mediators including cytokines, such as IL1-α, IL-β, IL-8, TNF-α and INF-α [25,26,27,28]. H. Duan et al. [29] detected IL-8 positive neutrophils in psoriatic lesions by using an immunohistochemical technique as a second source of IL-8 in psoriasis. The first described cells of psoriatic plaque capable of releasing IL-8 were keratinocytes [30]. Moreover, IL-8 production by neutrophils in response to stimulation with LPS or TNF-α was confirmed in psoriasis patients [29]. Also, the secretion of IL-8 by fibroblasts was observed by other authors [19,31,32]. In our experiment the secretion of IL-8 increased after stimulation with TNF-α. It is worth noting that lower secretion was observed for FPPL fibroblasts. This observation may suggest an active involvement of these fibroblasts in inflammatory response in vivo. Neutrophilsfibroblasts interaction (particularly FPL-NPV) caused an increase in the secretion of IL-8.

The inflammation in psoriasis is primarily biased towards the Th1 phenotype with the overexpression of INF-γ, TNF-α, IL-1 and IL-2. Th1 pathway is stimulated by IL-12 and, therefore, this cytokine may be central to the initiation and promotion of the psoriatic process. In fact IL-12 mRNA and protein levels are elevated in psoriatic lesions when compared with normal and nonlesional psoriatic skin. The biologically active IL-12 p70 heterodimer is thought to be strongly expressed in psoriatic skin lesions and mainly found on mononuclear cells and some neutrophils in the upper dermis, sparing the epidermis [33]. We assessed the secretion of IL-12 by neutrophils of peripheral blood in normal controls and psoriatic patients and by fibroblasts of healthy donors and psoriatic patients. The secretion of IL-12 by neutrophils was lower in patients with psoriasis. Fibroblasts did not secrete IL-12 but fibroblastsneutrophils interaction caused an increase in secretion of IL-12, however stimulation with IL-8 and TNF-α did not exert any effect on production of IL-12. These results may suggest that TNF-α and IL-8 do not influence the secretion of IL-12 by neutrophils. The increased secretion as a result of fibroblastsneutrophils interaction shows the importance of cells cooperation. Moreover, fibroblasts-neutrophils cooperation might be additional source of IL-12 which plays a central role in the differentiation of Th1. In contrast to the overexpression of proinflammatory cytokines, a relatively low level of the expression of antiinflammatory IL-1RA and IL-10 cytokines has been found. It may suggest an insufficient counterregulatory capacity in psoriasis which might have a genetic background [34,35]. IL-10 anti-inflammmatory cytokine is absent or is expressed at low levels in psoriasis. Moreover, there is no information available regarding the IL-10 expression by psoriatic peripheral blood leukocytes. IL-10 mRNA was detected in almost all skin punch biopsies taken from patients with psoriasis. Low levels were found in uninvolved and psoriatic skin. IL-10 secretion by peripheral blood leukocytes in psoriatic patients was lower when compared with patients with other dermatosis and healthy donors. Ksuru Asadullah et al. used IL-10 as a potential antipsoriatic medicine. The authors hypothesize that the antipsoriatic activity of IL-10 results from effects on different cell populations including T cells, APCs and keratinocytes and their interactions [36]. We assessed the secretion of IL-10 by neutrophils and fibroblasts of normal controls and psoriatic patients. We observed IL-10 release only by neutrophils of normal controls and this secretion increased after stimulation with IL-8 and TNF. Fibroblasts did not secrete IL-10 in basal conditions and after treatment with IL-8 and TNF. Fibroblast-neutrophil interaction intensified IL-10 release by neutrophils but only in normal controls. After stimulation with TNF and IL-8, the secretion of IL-10 did not increase. Our results partly confirm previous observations [34,36] and additionally may suggest that fibroblasts are not a source of IL-10.

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Our short experiment joins in the discussion about network cytokines and cells participating in maintenance of inflammatory process in psoriasis. Neutrophils are present at every stage of the disease. There may be two mechanisms of the accumulation of neutrophils: local autocrine (neutrophils may perpetuate their own recruitment by releasing IL-8 by themselves, migrating from circulation through the papillary dermis into the epidermis) and local paracrine (keratinocyte-derived IL-8 may initiate the first step of trafficking of neutrophils into the epidermis). Terui et al. suggest that PMNs are capable of potentiating the T-cellassociated inflammation-sustaining loop by activating both epidermal keratinocytes and T cells through the production of cytokines, active oxygen intermediates and proteolytic enzymes, and the expression of HLA-DR on their surfaces and propose a neutrophil-associated inflammation-boosting loop as an inducer of “acute” inflammation with focal infiltration of PMNs in psoriatic lesions [37]. Our results confirm another source of IL-8 and may suggest another way of the maintenance of inflammatory reaction shared by neutrophils and fibroblasts and participation neutrophils and fibroblast in switch from acute to chronic inflammation.

CONCLUSIONS On the whole, we can say that fibroblasts are a source of inflammatory cytokines (particularly IL-6 and IL-8) and their secretion is modulated by different stimuli [10] and our observations are in accordance with others [20,24,29]. It’s worth noting that fibroblasts via cytokines or direct interaction may cooperate with neutrophils in the maintenance of inflammation in psoriasis and probably do not exert any antiinflammatory effect.

ACKNOWLEDGEMENTS This study was supported by the Grant from the Scientific Council in Warsaw (no 4PO5A 07918) and Medical University of Lodz Grants (no 502-11-726 and no 503-1019-1). The authors would like to express great thanks to Ms. Bogumila Kolago and Ms. Aleksandra Slusarczyk for the preparation of fibroblast cultures.

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IL-8, IL-12 and IL-10 cytokines generation by neutrophils, fibroblasts and neutrophils- fibroblasts interaction in psoriasis

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